DNA Fragments for forming plasmids

ABSTRACT

The present invention relates to novel, broad bacterial host range small plasmid deoxyribonucleic acid rings which serve as cloning vehicles for DNA fragments, particularly those separated from other plasmid rings or from chromosones, recombined with the small plasmid rings and to the processes for recombining the plasmid rings and to processes for transferring them between host bacteria. In particular, the present invention relates to the aggregate plasmid ring RP1/pRO1600, to pRO1600 and plasmid ring derivatives thereof, particularly including pRO1601; pRO1613 and pRO1614, all of which are carried for reference purposes in Pseudomonas aeruginosa ATCC 15692 (also known as strain PAO1c) and are on deposit at the Northern Regional Research Laboratories (NRRL) of the United States Department of Agriculture at Peoria, Ill. The plasmid ring RP1 (also known as R1822) is deposited in Pseudomonas aeruginosa NRRL-B-12123 (and is a known plasmid ring). The pRO1600 portion of the aggregate is a new plasmid ring. The novel small plasmid rings are particularly useful for recombinant genetic manipulation wherein the DNA fragments are introduced into the plasmid rings to produce useful, cloneable characteristics in the host bacterium, particularly chemical generating characteristics.

This application is a division of application Ser. No. 147,563, filedMay 8, 1980.

SUMMARY OF THE INVENTION

The present invention relates to novel small plasmid rings having abroad bacterial host range and to the processes for recombining thefragments with other DNA fragments in vivo or in vitro and/or fortransferring the plasmids between host bacteria. In particular, thepresent invention relates to plasmid rings which are derivatives of theaggregate plasmid rings RPl/pRO1600 which occurred by chance only onceas a result of a mutation in Pseudomonas aeruginosa ATCC 15692 duringconjugation of the bacteria containing the natural plasmid ring RPl(also known as R1822). The novel plasmids are referred to herein aspRO16xy wherein x and y are integers.

PRIOR ART

The prior art commercial efforts involving recombinant geneticmanipulation of plasmids for producing various chemicals have centeredon Escherichia coli as a host organism. The reason is that therecombinant plasmids are not compatible with other host organisms.However, E. coli is not the most desirable organism to use for thesepurposes because of concerns about its ability to grow in the intestinaltract in mammals and also because of its ability to produce disease. Itwould be very desirable to be able to use host bacteria besides E. coliwith plasmids having a broad bacterial host range. The present inventionis concerned with such plasmids. It is particularly concerned with theuse of organisms that will not survive at mammalian body temperatures.

The following are definitions used in reference to the presentinvention:

The phrase "bacterial conjugation" means the mating and genetic transferbetween at least two bacterial hosts, the donor being designated as"male" and the recipient as "female".

The phrase "plasmid aggregate" means an association between two plasmidrings wherein each plasmid maintains its structure as a ring and whereineach ring is capable of separate recombinant genetic manipulation.

The term "transport" means any process whereby DNA is transferred fromone bacterium to another.

The term "transduction" means DNA plasmids or fragments caused to betransported by bacterial viruses from one bacterium to another in vivoby natural processes.

The term "transformation" means the injection of DNA stripped from awhole bacterial cell in vitro into a host bacterium.

The term "transposition" means the movement of genetic material from oneportion of a DNA molecule to another or from one DNA molecule toanother.

The term "transposon" refers to the genetic material transferred bytransposition.

The phrases "DNA source" or "DNA fragment" means DNA from chromosomal orextrachromosomal cellular elements (such as plasmids) derived fromeucaryotic or procaryotic cells and/or their parasites includingviruses.

The state of the patent art is generally described in U.S. Pat. Nos.3,813,316; 4,038,143; 4,080,261; 4,082,613 and 4,190,495. These patentsdescribe prior art processes related to the present invention.

Two elements have been identified as necessary for the replication of aDNA molecule in vivo other than the battery of enzymes and proteinsrequired for synthesis of the DNA molecule per se. They are (1) a regionon the DNA molecule which is identified as the origin of DNA replicationand which is recognized by a protein and also (2) the gene for thisprotein which is found on the same DNA molecule. Given this minimumspecification for a DNA molecule capable of autonomous self-reproductionin a bacterial cell, additional pieces of DNA may be included insofar asthey do not interfere with the two functions identified above. Thecombination of 1 and 2 above found in nature contained within andmaintaned extrachromosomally by host bacterial species has been calledplasmids or extrachromosomal elements (Bacteriological Reviews,September, pp. 552-590 (1976)). Until recently, microbial geneticistshave manipulated DNA fragments in vivo using host bacterial cellrecombination mechanisms to recombine DNA from the bacterial chromosomeinto plasmids or to effect recombination between plasmids co-maintainedin the same host bacterium. This practice has allowed purification ofvarious regions of a more complex genome by removal of a portion of thecomplex genome to another replicating unit. However, this practice doesnot normally allow for the construction of plasmid-hybrids comprised ofDNA from disparate sources or transported across large biologicalbarriers from different organisms.

In recent years, however, techniques for the in vitro manipulation andrecombination of heterogeneous fragments of DNA have allowed theconstruction of hybrid DNA molecules. A brief summary of the in vitroprocess quoted from "Recombinant Molecules: Impact on Science andSociety" R. F. Beers and E. G. Bassett, eds., pp 9-20, Raven Press, NewYork (1977)) follows.

"There are several important technical components to in vitrorecombinant (DNA) technology which ultimately result in the insertion ofDNA fragments from any source into replicons (plasmids)--and theirrecovery as replicating elements in bacteria. These components are:

1. the systematic dissection of the DNA molecules of interest withrestriction endonucleases (see Roberts, in Recombinant Molecules, for adiscussion of these enzymes);

2. the rejoining of DNA fragments by ligation to an appropriate cloningvehicle (or replicon);

3. the transformation of a cell with the recombinant DNA and selectionof cells containing the recombinant plasmid; and

4. the identification and characterization of the resulting "cloned"fragment of DNA."

This invention relates to all of the above, particularly item 2, namely,the requirement for an appropriate cloning vehicle (plasmid) and thenovel properties of the plasmid cloning vector described hereincontributing to the utility of the transformation process describedabove.

Biological properties of a plasmid potentially useful for molecular DNAcloning have been summarized by D. R. Helinski et al, in "RecombinantMolecules: Impact on Science and Society, p. 151, R. F. Beers and E. G.Bassett, eds., Raven Press, New York (1977)).

"Since the initial demonstration of the utility of a plasmid element forthe cloning of genes in Escherichia coli--a variety of plasmid elementshave been developed as cloning vehicles in both E. coli and (fordifferent plasmids) in other bacteria. These newer cloning vehiclespossess the following plasmid properties that are advantageous for thecloning of DNA:

1. stable maintenance in the host bacterial cell;

2. non-self-transmissibility;

3. ease of genetic manipulation;

4. ease of isolation;

5. the capacity of joining with and replicating foreign DNA of a broadsize range;

6. ease of introduction of the in vitro generated hybrid plasmid into abacterial cell."

The foregoing specifications relating to characteristics considereddesirable for the maximum utility of a plasmid useful for recombinantDNA technology, however, do not include a consideration for enhancedutility of a cloning plasmid with a broad bacterial host range.

Most bacterial plasmids described to date can be maintained only inbacterial species closely related to the bacterium from which theplasmid has originally been isolated. Because of this, the requirementsassociated with the maintenance and duplication of the particularplasmid's DNA generally are specific to the plasmid in question. Therehave been, however, exceptions to this general observation. For example,Olsen (the inventor herein) and Shipley (Journal of Bacteriology, 113,No. 2, pp. 772-780 (1973)) showed that a plasmid specifying multipleantibiotic resistances, designated R1822 (and later changed to RP1), wastransferred to a variety of bacterial species representative of relatedand unrelated bacterial genera by sexual conjugation. The origin of thestrain Pseudomonas aeruginosa 1822 from which RPl was later obtained isset forth in Lowbury, E. J. et al. Lancet ii 448-452 (1969). Thebacterial host range of the plasmid RPl includes Enterobacteriaceae,soil saprophytic bacteria (Pseudomonas), Neisseria perflava, andphotosynthetic bacteria. Plasmid RPl, then, is an example of a broadhost range bacterial plasmid which freely transfers among unrelatedbacterial species.

The plasmid ring RPl is relatively large. The large size and compositionof this plasmid ring makes the process of bacterial transformationinefficient. It would be a significant improvement in the art to providea small plasmid ring as a cloning vehicle and recombinants thereof whichwere easily and widely transportable particularly by transformation,from bacterial host to bacterial host. The plasmids would beparticularly useful if they included a single phenotypic marker forantibiotic resistance for identification purposes. It would also be animprovement to provide processes for in vivo or in vitro recombinationof fragments of the small plasmid ring with other genetic fragments.

OBJECTS

It is therefore an object of the present invention to provide novelplasmid fragmentation and recombination and/or transport processes.Further it is an object to provide novel recombinant plasmid ringsderived from small plasmids which act as cloning vehicles when combinedin the recombinant plasmid rings and wherein the plasmid rings have abroad bacterial host transfer range. It is particularly an object of thepresent invention to provide recombinant plasmid rings which have usefulchemical generating properties or some other useful characteristic inthe host bacterium. These and other objects will become increasinglyapparent by reference to the following description and the drawings.

IN THE DRAWINGS

FIG. 1 shows slab agarose gel electrophoresis patterns for RP1 of theprior art (A), E. coli V517 electrophoresis size standard (D) and forRPl/pRO1600 (B) and other plasmids (C, E, F) of the present invention.The longer the pattern, the smaller the plasmid.

FIG. 2 shows restriction endonuclease PstI and BglI maps in megadaltons(daltons×10⁶) for the preferred plasmids of the present invention,particularly plasmids pRO1601, pRO1613, pRO1614 and pRO1600. Numericalvalues in parenthesis represent molecular size in daltons×10⁶ forrestriction endonuclease fragments. Numerical values above or below theunbroken horizontal line are map distance of the restrictionendonuclease recognition site in daltons×10⁶ from zero as defined by thesingle PstI restriction endonuclease-DNA cleavage site present inplasmid pRO1600.

FIG. 3 shows slab agarose gel electrophoresis patterns for: pRO1614 (C);DNA from E. coli; V517 of the prior art for reference purposes (B) andDNA from transformant clones capable of growth without L-isoleucine orL-valine (A), referred to as pRO1615, or L-methionene (D), referred toas pRO1616, as a result of the recombinant modification of pRO1614.

GENERAL DESCRIPTION

The present invention relates to a recombinant deoxyribonucleic acidplasmid ring including a first plasmid fragment, the first plasmid beingoriginally derived from a plasmid aggregation with plasmid RPl andmeasuring about 2×10⁶ daltons or less in molecular size and having acritical restriction endonuclease BglI digestion fragment measuring0.83×10⁶ daltons in molecular size which is indispensible forreplication, covalently combined with at least one seconddeoxyribonucleic acid fragment which is a restriction endonucleasedigestion fragment ligated to the first plasmid in vitro or a naturallyoccurring fragment inserted by a bacterium in vivo into the firstplasmid as a recombinant plasmid ring capable of being carried byPseudomonas aeruginosa PAO ATCC 15692 (PAO1c) and having a broadbacterial host transmission range, wherein the second fragmentcontributes a useful chemical characteristic to the recombinant plasmidring and wherein the plasmid ring clones itself by DNA replicationduring cell devision of the host bacterium.

The present invention also relates to the bacterial composition whichcomprises a deoxyribonucleic acid plasmid ring including a first plasmidfragment, the first plasmid being originally derived as a plasmidaggregation with plasmid RPl measuring about 2×10⁶ daltons or less inmolecular size and having a critical restriction endonuclease BglIdigestion fragment measuring 0.83×10⁶ daltons in molecular size which isindispensible for replication, covalently combined with at least onesecond deoxyribonucleic acid fragment which is a restrictionendonuclease digestion fragment, ligated to the first plasmid in vitroor a naturally occurring fragment inserted by a bacterium in vivo intothe first plasmid as a recombinant plasmid ring capable of being carriedby Pseudomonas aeruginosa PAO ATCC 15692 (PAO1c) and having a broadbacterial host transmission range, wherein the second fragmentcontributes a useful chemical characteristic to the recombinant plasmidring and wherein the plasmid ring clones itself by DNA replicationduring cell division of the host bacterium; and a host bacterium.

The present invention also relates to a process for transporting DNAplasmids to bacterial hosts in vivo using the processes of bacterialconjugation, transformation or transduction, the improvement whichcomprises transporting a deoxyribonucleic acid plasmid ring, including afirst plasmid ring originally derived as a plasmid aggregation withplasmid RPl measuring about 2×10⁶ daltons or less in molecular size andhaving a critical BglI restriction endonuclease digestion fragmentmeasuring 0.83×10⁶ daltons in molecular size which is indispensible forreplication alone or with a fragment from the first plasmid ring,covalently combined with at least one second deoxyribonucleic acidfragment which is a restriction endonuclease digestion deoxyribonucleicacid fragment ligated to the first plasmid or a naturally occurringfragment inserted by a bacterium in vivo in the first plasmid to form arecombinant plasmid ring, wherein the plasmids are capable of beingcarried by Pseudomonas aeruginosa PAO ATCC 15692 (PAO1c), wherein theplasmids have a broad bacterial host range, and wherein the plasmidclones itself by DNA replication during cell division of the hostbacterium.

The present invention relates to the process which comprises providingan aggregate of a first plasmid with a second plasmid, wherein thesecond plasmid has a transposon which produces a useful chemicalcharacteristic and wherein the first plasmid was originally derived asan aggregation with RPl and measures about 2×10⁶ daltons or less inmolecular size and has a critical BglI endonuclease digestion fragmentmeasuring 0.83×10⁶ daltons in molecular size indispensible forreplication; providing the aggregate plasmid in a plasmid receptivebacterial cell; growing the receptive bacterial cell in a growth mediumwith the aggregate plasmid to randomly produce a recombined plasmidincluding the transposon and first plasmid, wherein the recombinedplasmid replicates upon division of the bacterial cell; and selectingthe bacterial cells with the recombined plasmid with the transposon.

Further, the present invention relates to the process for producingrecombinant deoxyribonucleic acid plasmids which comprises providing afirst plasmid and a second deoxyribonucleic acid source, wherein thefirst plasmid was originally derived from a plasmid aggregation withplasmid RPl measures about 2×10⁶ daltons or less in molecular size andhas a critical restriction endonuclease BglI digestion segment measuring0.83×10⁶ daltons in molecular size which is indispensible forreplication; reacting the plasmid and the second deoxyribonucleic acidsource with at least one restriction endonuclease which cleaves thefirst plasmid and the second deoxyribonucleic acid source into linearDNA fragments; and randomly recombining the linear deoxyribonucleic acidfragments using ligation to form recombinant plasmids which replicate ina bacterial cell.

Finally, the present invention relates to a deoxyribonucleic acidfragment for forming plasmids, the fragment being formed from a firstplasmid originally derived from a plasmid aggregation with plasmid RPland measuring about 2×10⁶ daltons or less in molecular size, wherein thefragment has a critical restriction endonuclease BglI digestion fragmentmeasuring 0.83×10⁶ daltons in molecular size which is indispensible forreplication in a plasmid. BglI was isolated from Bacillus globiggi. BglIproduces 5' termini as follows: ##STR1##

The plasmid content of bacteria can be conveniently and expeditiouslyestimated by employing the technique of slab agarose gel electrophoresiswith visualization of the result on photographs of the resultingelectropherogram (for example, see Hansen and Olsen, Journal ofBacteriology, 135, No. 1, pp. 227-238 (1978)). Thus DNA waselectrophoresed as shown in FIG. 1 as follows: A, DNA extracted fromPseudomonas aeruginosa NRRL 12123; B, DNA from RPl/pRO1600; C, DNA fromprRO1601; D, DNA from Escherichia coli V517, a multi-plasmid-containingstrain used as a size standard (Plasmid, 1, pp. 417-420 (1978)); E, DNAfrom pRO1613; F, DNA from pRO1614. This procedure also allows anestimate of the molecular size of any plasmids present when suitablestandards are incorporated into the procedure.

When the plasmid RPl is caused to transfer from one bacterium to anotherby the process called bacterial conjugation (or sexual mating), therecipient bacterium that has newly acquired the plasmid normallycontains a plasmid that is indistiguishable on electropherograms fromthe plasmid present in the donor bacterium. In FIG. 1, file A, isdepicted the usual appearance of plasmid RPl when extracted from donorcell populations or a recipient cell population subsequent to itstransfer. On one occasion another result was obtained. Analysis of aculture derived from a single recipient cell of ATCC 15692 (strain PAO2)which had received plasmid RPl in a mating experiment with Pseudomonasaeruginosa PAO25 (another mutant variation of ATCC 15692, the same asPAO2 as described above except that it requires leucine and arginine forgrowth and maintenance and on deposit at the University of Michigan)produced a variant plasmid aggregate. The transconjugant showed thepresence of not only the parent plasmid, but also a second andconsiderably smaller plasmid (FIG. 1, file B). This result has not beenseen again after many repetitions of the process. The parental-sizeplasmid is shown at the top of file B; the anomolous smaller plasmidappears at the lower portion of the electropherogram depicted in FIG. 1.The size of the lower plasmid, estimated by comparison and calculationrelative to the size standards in file D, is 2×10⁶ daltons compared to38×10⁶ daltons for RPl. The appearance of the small plasmid, then,reflects a novel event which, in general terms, may be considered arandom mutational event which most likely occurred during the transferto the recipient bacterium of the parental plasmid, RPl. I havedesignated the small plasmid pRO1600 and the aggregation with RPl asRPl/pRO1600.

The utility of a small plasmid for application in recombinant DNAtechnology derives, in part, from its ability to encode geneticinformation for a unique metabolic trait not possessed by the hostbacterium. Accordingly, the presence or absence of the plasmid,subsequent to genetic manipulations, can be determined on the basis ofthe presence or absence of the metabolic trait in question. Preliminaryanalysis of the bacterial strain shown in FIG. 1, file B showed nounique metabolic trait (phenotypic character) associated with thepresence of plasmid pRO1600. I therefore applied standard bacterialgenetic techniques to add a distinctive phenotypic trait to pRO1600allowing its detection in later experiments whereby I tested its abilityto be transformed from partially purified DNA solutions to recipientbacterial strains. The distinctive phenotypic trait added was a piece ofDNA which encodes genetic information for resistance to the antibiotic,carbenicillin. Accordingly, all bacteria maintaining plasmid pRO1600with this piece of DNA will grow in the presence of carbenicillin unlikethe parental, plasmid-free, bacterial strain.

The genetic trait for carbenicillin resistance was added to plasmidpRO1600 by the genetic process called transposition. Some antibioticresistance genes, called transposons, are able to move from one locationto another on a piece of DNA or alternatively, able to move from one DNAmolecule to another within the bacterial cell by the genetic processcalled transposition (S. Cohen, "Transposable genetic elements andplasmid evolution" Nature, 263, pp. 731-738 (1976)). These geneticelements accomplish this process in the absence of bacterial hostgenetic recombinational mechanisms. Plasmid RPl, shown in file A, FIG.1, contains a transposon called Tnl which encodes genetic informationfor resistance to carbenicillian and related antibiotics (penicillin,ampicillin). Transposon, Tnl is a transposable genetic element of3.2×10⁶ daltons molecular size. Accordingly, DNA molecules that havebeen transposed by Tnl would increase in size by 3.2×10⁶ daltons.

Transposition occurs randomly in populations of bacterial cells whichall contain a donor DNA molecule which has the transposon. Thus, in thecase of bacterial strain Pseudomonas aeruginosa RPl/pRO1600, one wouldexpect a small proportion of the bacterial cells to containtransposed-variant plasmids. For example, as shown in file B, FIG. 1, afew bacterial cells in the culture would contain a DNA molecule largerby 3.2×10⁶ daltons than the small DNA molecule shown at the bottom ofthe electropherogram (the plasmid designated pRO1600.) The relativelysmall number of these bacteria in the culture, however, precludes theirdetection on agarose gels as shown in FIG. 1. However, these transposedderivatives of plasmid pRO1600 can be detected and isolated by using thegenetic technique called bacterial transformation. By this process, DNAthat has been extracted from cells called donors is added to aplasmid-free recipient bacterial cell culture. The admixture, in turn,is then grown on bacteriological medium containing, in this instance,the antibiotic carbenicillin. Under these conditions, only bacterialcells that have taken up and replicated a genetic element which encodesgenetic information for the antibiotic carbenicillin will grow to densepopulations. When this experiment is done, two types of carbenicillinresistant bacterial strains would be expected: those which have receivedall of the donor parent plasmid, RPl; those which have only receivedpRO1600 combined with the transposon which encodes carbenicillinresistance, Tnl, which is now contained within its structure. The formerclass of possible bacterial isolates can be distinguished by the factthat these cells will be resistant to not only carbenicillin, but alsotetracycline and kanamycin, other genes which are part of the structureof RPl. In the case of the latter possibility; however, these bacterialisolates would only demonstrate resistance to the antibiotic,carbenicillin, since they have only received Tnl which has jumped fromplasmid RPl to plasmid pRO1600 co-maintained at the same time in abacterial cell. Such a bacterial strain was obtained by the processdescribed above and its DNA, following extraction of its plasmid andelectrophoresis is shown in file C, FIG. 1. This plasmid, designatedpRO1601, shows slower electrophoretic mobility than plasmid pRO1600reflecting its larger size. When calculations are done using thestandard DNA depicted in file D, FIG. 1, it is determined that pRO1601is larger than pRO1600 by 3.2×10⁶ daltons molecular size. Thisrelationship, then, suggests that Tnl has been added to pRO1600 andaccordingly a unique metabolic trait (phenotypic marker) has been addedto pRO1600 allowing its detection by testing for resistance to theantibiotic, carbenicillin.

DNA molecules are linear polymers comprised of substituent moleculescalled purines and pyrimidines. The exact linear sequence of joinedpurines or pyrimidines, then, defines any region of a DNA molecule ofinterest. Analagous regions of any DNA molecule can be detected by theircleavage with unique enzymes specific in their activity on those regionscalled Class II restriction endonucleases (B. Allet, "Fragments producedby cleavage of lambda deoxyribonucleic acid with Haemophilusparainfluenzae restriction enzyme HpaII" Biochemistry, 12, pp. 3972-3977(1972)). The specific regions of a DNA molecule, called recognitionsequences, which serve as sites for cleavage by any of more than 40specific restriction endonucleases will be distributed differently orabsent when DNA from different biological sources is compared.Conversely, related or identical DNA molecules will yield an identicalset of fragments, subsequent to restriction endonuclease digestion andanalysis by the techniques of slab agarose gel electrophoresis.Therefore new plasmids can be produced subsequent to digestion asdescribed above. If the size of the DNA molecule is small, and thenumber of fragments generated by treatment with restriction endonucleaseenzyme is few, these fragments can randomly associate in different orderthan originally present on the parent molecule. Following this randomre-association, the fragments can be again covalently joined when there-associated complex is treated with a second enzyme called DNA ligaseand required co-factors. The foregoing process, then, can be applied tothe analysis of structure of DNA molecules and production of newcombinations of DNA sequences. By the foregoing process, then, plasmidstructure can be altered by deleting non-essential regions of DNA or theproduction of a DNA molecule comprised of DNA from biologicallyunrelated sources.

The present invention particularly relates to plasmid pRO1601, itsmodification and its utility for genetic cloning and novelty by applyingthe rationale and procedures described in general terms above andcommonly referred to as recombinant DNA technology. The essentialfeatures of this recombinant DNA technology have been summarizedpreviously (S. Cohen, Scientific American, July, pp. 25-33 (1975)).

Analysis of plasmid pRO1601 DNA (restriction endonuclease mapping) byconventional procedures referenced above produced the structure depictedin FIG. 2, part A. Inspection of this genetic map shows the presence offour restriction endonuclease recognition sites for the restrictionendonuclease, PstI. Transposon, Tnl, which was added to pRO1600 toproduce pRO1601 is known to contain 3 PstI restriction endonucleasesites (J. Grinsted et al., Plasmid, 1, pp 34-37 (1977)). Therefore, thepRO1600 region of the recombinant plasmid that has undergonetransposition by Tnl must contain a single PstI site. Also, since thesize of the PstI fragments associated with Tnl has been estimated (seeabove ref.), the particular site for PstI cleavage which is unique topRO1600 can be identified. This site has been chosen as the referencesite for mapping and appears at the left of the drawing shown in part A,FIG. 2. Other sites, representing regions of the recombinant plasmid,pRO1601, which are part of Tnl, have been juxtaposed in relation to thepRO1600 PstI site. Also shown on this genetic map are specificrecognition sites for the restriction endonucleases BglI. These weredetermined by comparing other variants of Tnl-transposed plasmid pRO1600which differed from pRO1601 with respect to the point of the insertionof the transposon Tnl into pRO1600. For this, conventional proceduresattendant to recombinant DNA technology were used.

The PstI restriction endonuclease site drawn at 1.21 on panel A, FIG. 2which is known to be part of the region reflecting the addition of thetransposon, Tnl, is known to be in the middle of the genetic regionwhich encodes for resistance to carbenicillin. Accordingly, insertion ofextra DNA at this point will destroy the contiguity of the DNA sequenceand result in the loss of the ability of this region of the DNA tospecify the enzyme associated with carbenicillin resistance. Todemonstrate the utility of pRO1601 for molecular cloning, a piece of DNAwas inserted into this site. This piece of DNA, however, containsgenetic information for resistance to the antibiotic tetracycline.Consequently, if this piece were incorporated into plasmid pRO1601 atthis site, ligated to form a closed circular DNA structure, transformedinto a recipient bacterium and cultured with selection for the abilityto grow in the presence of tetracycline, cloning of the insertedfragment would have occurred.

The source of DNA for these experiments was yet another plasmid calledpBR322 (F. Bolivar et al., "Construction and Characterization of newcloning vehicles II. a multipurpose cloning system" Gene, 2, pp. 95-13(1977)). Plasmid pBR322, typical of cloning plasmids developed to date,is unable to be maintained in bacteria not closely related to thebacterium of its origin, Escherichia coli. Consequently, when thisplamid DNA is introduced into an unrelated bacterium, Pseudomonasaeruginosa, it will not confer the ability to grow in the presence ofantibiotics for which it encodes resistance, namely, resistance tocarbenicillin and tetracycline. If, on the other hand, plasmid pBR322DNA becomes part of the structure, by recombination, of a plasmid suchas plasmid pRO1601, its genetic functions will be conferred to therecombinant plasmid-containing pRO1601-pBR322 hybrid. Plasmid pBR322,analagous to plasmid pRO1601, also contains a gene specifying resistanceto the antibiotic, carbenicillin. This gene too has a site forrestriction endonuclease PstI within the region of its DNA associatedwith carbenicillin resistance. To test the assumption that part of thecarbenicillin resistance gene of plasmid pRO1601 could be matched upwith part of the analogous gene from plasmid pBR322 resulting, in thiscase, in the conservation of carbenicillin resistance for a hybridmolecule joined in one place at this juncture, I cleaved both plasmidpRO1601 and plasmid pBR322 DNA with the restriction endonuclease PstI.This produces two linear molecules from the parent circular structureswhich can randomly associate; one of four possible pieces from plasmidpRO1601 and a linear single piece of DNA derived from plasmid pBR322.Following the application of recombinant DNA technology, namely, plasmidcleavage, ligation and transformation with selection for the acquisitionof resistance to tetracycline, two groups of recombinant plasmids wereobtained as judged later by mapping with restriction endonucleases.These structures are shown in FIG. 2, panels B and C. They have beendesignated, respectively, pRO1613 and pRO1614.

The structure determined for plasmid pRO1613 conforms to that expectedfrom the following possibilities: reassociation of plasmid pRO1601 PstIfragments depicted on FIG. 2, panel A as traversing the distance between0 and 1.21 and between 1.21 and 2.93. However, theoretically, thisproduces a plasmid having two PstI sites not one as shown on FIG. 2,Panel B, for the resultant plasmid, pRO1613. Therefore, it is probable,based on precedent from other systems, that the DNA at 0 and 2.93 on thepRO1601 map has been altered by unknown factors during the process whicheffected the deletion of a PstI site but still allowed reformation of aclosed ring structure required for survival of the plasmid by abacterium and its progeny cells during growth and reproduction of thebacterial culture. Plasmid pRO1613 has potential utility for the cloningof restriction endonuclease PstI generated fragments of DNA whosepresence can be ascertained by the embodiment within the cloned piece ofDNA of a metabolic trait for which recombinant progeny can be selected.

The structure determined for plasmid pRO1614 conforms to that expectedfor a hybrid-recombinant plasmid which contains a part of the cloningvector plasmid, pROFIG. and the cloned fragment, in this case, plasmidpBR322. Furthermore, the formation of this recombinant plasmid, pRO1614,and its transformation into Pseudomonas aeruginosa is accompanied by theoccurrence of bacteria which are now both carbenicillin resistant andtetracycline resistant. Clearly, then, as hypothesized above, part ofthe carbenicillin resistance gene has been derived from plasmid pRO1601(region to the left of the PstI site shown at 1.21) and part of thecarbenicillin gene has been derived from the inserted DNA fragment(region to the right of 1.21, FIG. 2, Panel C,) which is part of plasmidpBR322. This result, then, has demonstrated the utility of plasmidpRO1601 for cloning unrelated DNA (plasmid pBR322). This result alsodemonstrates the derivation of related, albeit size-reduced, derivativeplasmids of greater utility than the parent plasmid DNA molecule,pRO1601.

Additional information regarding the essential features of plasmidpRO1601 required for maintenance by a host bacterium is also providedhere by these experiments: the region on FIG. 2, Panels A, B and C from0.05 to 0.88 map units, measuring 0.83 megadaltons is common to allplasmids shown. Accordingly, this is the region of plasmid DNA presentoriginally on plasmid pRO1600 which is essential for the replication andmaintenance of the plasmid cloning vector and its possible derivatives.This region, defined by its restriction endonuclease restriction enzymesites at 0.05 and 0.88 map units is a critical embodiment of thisinvention.

In addition to the foregoing, restriction endonuclease digest analysisof plasmids described in the development of the invention allowdeduction of the genetic map of plasmid pRO1600 independent of RPl. Itsrestriction endonuclease digest map is shown in panel D, FIG. 2.

The present invention particularly relates to the process whichcomprises the addition of chromosome DNA fragments produced bychromosome cleavage with a restriction endonuclease into a broad hostrange replicator plasmid and its apertinent DNA which has also beencleaved by the same restriction endonuclease. This admixture of plasmidand chromosome DNA fragments is then followed by ligase treatment andtransformation of recombined DNA to a suitable bacterial host.

The source of DNA for this series of cloning experiments was plasmidpRO1614 and the Pseudomonas aeruginosa strain PAO chromosome. Thepurpose of these experiments was to demonstrate the utility of pRO1614for cloning chromosomal DNA fragments corresponding to genetic locationson the chromosome associated with specific metabolic functionscontributing to the biosynthetic activities of the bacterial cell. Forthis purpose a mutant of P. aeruginosa designated strain PAO236 (D. Haasand B. Holloway, Molecular and general genetics 144:251(1976)) was usedas the transformation-cloning recipient. Unlike the parent bacterial P.aeruginosa strain, PAO 1c (ATCC 15692), strain PAO236 has been mutatedto require the addition of the amino acids L-isoleucine, L-valine andL-methionine to growth medium to support cellular synthesis and growth.These nutritional requirements for the amino acids, then, allow for theselection of recombinant clones which contain chromosomal DNA fragmentsassociated with synthesis of these compounds: bacteria which havereceived the appropriate cloned DNA fragment acquire the ability to growin the absence of the amino acid for which its biosynthesis has mutatedto a requirement. It also follows from the application of this rationalethat the recombinant plasmid, in this instance pRO1614 plus the clonedchromosomal DNA fragment, will be larger than the molecular size ofplasmid pRO1614 reflecting the acquisition of additional DNA in themolecular cloning process. Within the DNA of plasmid pBR322 cloned intothe broad bacterial host range replicator plasmid, pRO1613, is containedthe genetic information which specifies resistance to the antibiotic,tetracycline. Within this region of the cloned fragment is also therecognition site for the restriction endonuclease. BamHl. It followsfrom this relationship, then, that interruption of the contiguity of thetetracycline resistance gene by the in vitro insertion of a cloned DNAfragment will destroy tetracycline resistance specified by this gene.Additionally, if the selection for cloned DNA fragments is on the basisof the acquisition of a specific metabolic trait, bacteria which havereceived the appropriate recombinant plasmid will grow in the absence ofthe metabolite corresponding to the specific metabolic trait inquestion. Such bacterial strains, were obtained by the process describedabove and their DNA, following extraction of their plasmids are shown inFIG. 3. In FIG. 3, file B contains reference DNA used as a sizestandard, file C shows plasmid pRO1614, file A shows plasmid pRO 1614with its DNA fragment now allowing growth of bacterial strain PAO236 inthe absence of the amino acids isoleucine and L-valine, referred to aspRO1615, and file D shows plasmid pRO1614 with its cloned DNA fragmentnow allowing growth of bacterial strain PAO236 in the absence of theamino acid L-methionine, referred to as pRO1616. Clearly, therecombinant plasmids shown in files A and D are larger than pRO1614.This is the expected relationship associated with the acquisition of aheterologous DNA fragment following the application of recombinant DNAtechonlogy.

SPECIFIC DESCRIPTION

1. Steps

The specific process for developing the genetic cloning plasmid into theform of an artificial, chemically derived, non-transmissible plasmid andits use for the genetic cloning of a representative fragment of DNA isas follows:

(1) Recognition of the novelty and potential usefulness of a mutantplasmid during routine analysis for the presence of the progenitorplasmid, RPl, subsequent to a genetic transfer experiment: (occurrenceof pRO1600);

(2) The addition of a distinctive phenotypic trait for resistance to theantibiotic, carbenicillin, to the small plasmid observed as an extraanamolous plasmid (production of plasmid pRO1601);

(3) Physical mapping of plasmid pRO1601 with restriction endonucleases:

(4) Size-reduction of plasmid pRO1601 (production of plasmid pRO1613),genetic cloning of plasmid pBR322 into plasmid pRO1601 (production ofplasmid pRO1614).

(5) Genetic cloning, using recombinant DNA technology, of bacterialchromosome DNA associated with growth by strain PAO236 in the absence ofL-isoleucine and L-valine or L-methionine.

2. Strains Used

Bacterial strains used in the development and characterization ofplasmids for genetic cloning and their saliant features are as follows:

(1) Pseudomonas aeruginosa PAO2. This bacterial strain is a mutant of P.aeruginosa 1c (ATCC No. 15692) which has been mutated to require theamino acid, serine, for its growth and maintenance;

(2) P. aeruginosa PAO2(RPl). This strain was derived from the foregoingbacterial strain PAO2 by the addition of plasmid RPl by the process ofbacterial conjugation from PAO 2 (RPl) NRRL-B-12123 previouslydescribed;

(3) Pseudomonas putida PPO131. This bacterial strain is a mutant of P.putida A.3.12 (ATCC no. 12633) which has been mutated to require theamino acid, histidine, for its growth and maintenance;

(4) Pseudomonas fluoroescens PFO141. This isolate was derived from awild-type strain isolated from nature. The clone of this strain wasinfluenced by its inability to grow and reproduce at temperaturesexceeding 32° C. Accordingly this physiological trait effectivelycontains growth of the bacterium and hence its plasmids to environmentsexcluding mammilian environments. P. fluorescens PFO141 is a mutant ofthe wild-type strain which requires the amino acid, histidine, forgrowth and maintenance.

(5) Escherichia coli ED8654. The strain was derived from E. coli K12 andrequires the amino acid, methionine, for its growth and maintenance. Ithas also been mutated to not restrict or modify DNA.

(6) Azotobacter vinelandii, AVM100. This is a strain isolated fromnature.

(7) Klebiella pneumoniae KPO100. This is a strain isolated from nature.

(8) Pseudomonas aeruginosa PAO236. This bacterial strain is a mutant ofP. aeruginosa 1c (ATCC No. 15692) which has been mutated to require theamino acids isoleucine, valine and methionine and other amino acids.

All of the cultures are maintained in the culture collection of theDepartment of Microbiology, University of Michigan Medical School andare freely available to qualified recipients. Such cultures are alsoavailable from other depositories.

3. Materials

The bacteriological medium used for routine maintenance and propagationof the above bacterial strains contained the following ingredients:

    ______________________________________                                        Bacto-tryptone         5 g                                                    Bacto-yeast extract    3.75 g                                                 Dextrose               1 g                                                    KNO.sub.3              4 g                                                    Distilled Water        1000 ml                                                ______________________________________                                    

When solid medium was used, Bacto-agar was added (15 g) prior tosterilization. Medium was sterilized by autoclaving at 121° C. for 15minutes. Antibiotic was added to the above medium after sterilizationand cooling to 50° C. The plasmid DNA was added to recipient bacterialcells and then selected for uptake and maintenance of the plasmid whichspecified resistance to an antibiotic. For carbenicillin it was 0.5 mgper ml; for tetracycline it was 0.025 mg per ml except for P. aeruginosaPAO2. For this bacterial strain, tetracycline was added at 0.05 mg perml medium.

4. Temperatures

Experiments utilizing P. putida or P. fluorescens were carried out at25° C. All other experiments were carried out at 37° C.

EXAMPLE 1

Addition of a selectable genetic marker to plasmid pRO1600 (productionof plasmid pRO1601).

For this example a partially purified DNA suspension derived from P.aeruginosa PAO2 (RPl) or from P. aeruginosa (RPl/pRO1600) was added to agrowing culture of P. aeruginosa PAO2. Following experimentalmanipulations attendant to the generally known process of bacterialtransformation, the admixture was deposited on the surface of solidmedium which contained the antibiotic, carbenicillin. This was incubatedat 37° C. for 24 hours and the number of bacterial colonies counted. Theresults of a typical experiment are shown below in Table 1. Thesecolonies, selected for the ability to grow in the presence ofcarbenicillin, were then sub-propagated (grown out) on nutrient mediumcontaining either the antibiotic tetracycline or the antibiotickanamycin to score for the co-nonselected acquisition of phenotypicmarkers other than carbenicillin resistance which were present in thebacteria used to prepare the transforming DNA.

                  TABLE 1                                                         ______________________________________                                                              Nonselected markers which                               Source of             Were Acquired by 100 Trans-                             Transforming                                                                            Number of   formant colonies                                        DNA       Transformants                                                                             Tetracycline                                                                              Kanamycin                                   ______________________________________                                        P. aeruginosa                                                                           190         100%        100%                                        PAO2 (RP1)                                                                    P. aeruginosa                                                                           397          92%         92%                                        (RP1)pRO/1600                                                                 ______________________________________                                    

The results shown in Table 1 indicate that 8 percent of thetransformants obtained from the P. aeruginosa PAO2 (RPl/pRO1600) DNAsuspension acquired resistance to carbenicillin only and not resistanceto tetracycline or kanamycin. This result, then, suggests that pRO1600with a Tnl transposon has been transformed as suggested in the foregoingbackground discussion. On the other hand, transformants derived from theuse of the P aeruginosa PAO2(RPl) DNA suspension, as expected, producedno transformants resistant to carbenicillin only, since this strain doesnot contain plasmid pRO1600 as a potential transposon acceptor plasmid.

EXAMPLE 2

Size-reduction of plasmid pRO1601 (production of plasmid pRO1613) andcloning of pBR322 DNA into plasmid pRO1601 (production of plasmidpRO1614).

For this experiment, partially purified DNA solutions identified in theTable 2 were treated with enzymes listed. Transformants were selectedusing P. aeruginosa PAO2 bacterial cells that had not previouslyreceived a plasmid. The antibiotic resistance(s) acquired by thebacteria as a consequence of their admixture with the various DNApreparations, treted as indicated, are shown on the right column of theTable.

                  TABLE 2                                                         ______________________________________                                                               Number                                                 Plasmid                of Trans-                                                                              Transformant                                  DNA Used Treatment of DNA                                                                            formants Phenotype                                     ______________________________________                                        pBR322   None             0     Not Relevant                                  pBR1601  None          69,000   carbenicillin                                                                 resistant                                     pRO1601  PstI digestion +                                                                               70    carbenicillin                                          ligation               resistant pRO1613                             pRO1601 +                                                                              PstI digestion +                                                                               2     carbenicillin                                 pBR322   ligation      resistant,                                                                       4     carbenicillin and                                                             tetracycline                                                                  resistant                                                                     pRO1614                                       ______________________________________                                    

Also noteworthy from the above experiments is the failure of plasmidpBR322, by itself to transform. Therefore, the expression of itstetracycline resistance, when cloned into plasmid pRO1601 reflects itsmaintenance by virtue of its association with critical functionsprovided by the vector, plasmid pRO1601.

EXAMPLE 3

Demonstration of broad bacterial host range for plasmids derived frompRO1600.

To demonstrate the utility of derivatives of plasmid pRO1600 (pRO1613,pRO1614) with respect to the usefulness of this invention for conductingcloning experiments using recombinant DNA technology in bacteria ofdisparate ecological niche and physiological properties, bacterialtransformation studies were done using bacteria other than P. aeruginosaPAO2 as the transformation-recipient bacterium. The results of typicalexperiments are shown below in Table 3. The procedure for producing andexacting the DNA is described in Hansen and Olsen referred to above.

For this work, approximately 0.5 micrograms of DNA suspended in 0.025 mlbuffer solution was added to approximately 1×10⁸ bacterial recipientcells in a volume of 0.2 ml. The solution was heat cycled between 0° and45° C. using a procedure described in Transformation of Salmonellatyphinium by Plasmid Deoxyribonucleic Acid, J. Bact. Vol 119 pp 1072 to1074 (1974) D. E. Lederburg and S. N. Cohen. This admixture, followingexperimental manipulation attendant to the known technique of bacterialtransformation, was deposited on the surface of solid medium whichcontained antibiotic. This was incubated at 37° C. for 48 hours at theappropriate temperature and the number of bacterial colonies counted.The results of these experiments are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Source of                                                                              Transformation-           Number of                                  Transforming                                                                           Recipient Bacterial                                                                          Selective  Transfor-                                  DNA      Strain         Antibiotic mants*                                     ______________________________________                                        pRO1613  P. aeruginosa PAO2                                                                           Carbenicillin                                                                            132,000                                    pRO1614  P. aeruginosa PAO2                                                                           Tetracycline                                                                              38,000                                    pRO1614  P. putida PP0131                                                                             Tetracycline                                                                                960                                     pRO1614  P. fluorescens PF0141                                                                        Tetracycline                                                                              24,600                                    pRO1614  E. coli ED8654 Tetracycline                                                                              5,500                                     pRO1614  K. pneumoniae KPM100                                                                         Tetracycline                                                                              1,300                                     pRO1613  A. vinlandii AVM100                                                                          Carbenicillin                                                                               176                                     ______________________________________                                         *out of 10.sup.8 potential recipient cells                               

The experiments shown above in Table 3, clearly indicate the broad hostrange feature of plasmids derived from plasmid pRO1600. Variation in theefficiency of the transformation process with the bacterial strain used,in all probability, is associated with the use of a transformationprocess which is not optimal for the bacterial strain in question.Accordingly, quantitative aspects of these results do not limit theutility of plasmid pRO1600-derivatives for genetic cloning experimentswith bacteria showing poor transformation efficiencies. In theseinstances, improvements in the process of bacterial transformation forthe particular bacterial strain in question should, in the future,enhance the efficiency of transformation per se using these bacterialstrains.

EXAMPLE 4

Demonstration of the molecular cloning of chromosomal DNA fromPseudomonas aeruginosa using the recombinant plasmid, pRO1614.

To demonstrate the utility of the derived plasmid, pRO1614, with respectto the usefulness of this invention for conducting cloning experimentsusing recombinant DNA technology in bacteria for the cloning ofbacterial chromosome genes, a cloning experiment was done for theselection and isolation of bacterial genes associated with thebiosynthesis of the amino acids L-isoleucine, L-valine and L-methionine.The procedure for producing chromosomal and plasmid DNA is described inHansen and Olsen referred to above.

For this work, approximately 0.5 micrograms of either chromosomal DNAextracted from PAO 1c or plasmid pRO1614 DNA were suspended in 0.025 mlbuffer and digested with the restriction endonuclease, BamHl. Theseparate solutions were then mixed and treated with the enzyme T4 ligaseand cofactors. This admixture, following experimental manipulationattendant to the technique of bacterial transformation, was deposited onthe surface of solid medium which was supplemented with the nutrientsrequired for growth by Pseudomonas aeruginosa for growth except in oneinstance the amino acids L-isoleucine and L-valine and in anotherinstance, the amino acid L-methionine. A single colony of bacterialgrowth appeared on each of the above selective medium. (A frequency ofone in 10⁸ recipients). When these recombinant DNA plasmids areextracted from transformation recipient bacteria, the resulting DNAshows high transformation frequencies when tested by retransformationinto yet another recipient. The frequency for amino acid biosynthesisacquisition corresponds to the frequency for the acquisition ofcarbenicillen resistance in these experiments. These colonies were thenpurified and grown up for the production of plasmid DNA.

FIG. 3 shows slab agarose gel electrophoresis portions for plasmid DNAfrom Pseudomonas aeruginosa PAO236. Bacterial cells were grown innutrient broth medium and plasmid DNA was extracted and processed asdescribed in reference to FIG. 1. DNA was electrophoresed and sampleswere as follows: A, DNA extracted from a transformant clone capable ofgrowth without L-isoleucine or L-valine (pRO1615); B, DNA fromEscherichia coli V517, a multi-plasmid-containing strain used as a sizestandard; C, DNA from Pseudomonas aeruginosa PAO2(pRO1614); D, DNAextracted from a transformant clone capable of growth withoutL-methionine (pRO1616). The estimated molecular sizes for therecombinant plasmids are 14×10⁶ daltons for the plasmid in file A;10.4×10⁶ daltons for the plasmid in file D.

Recombinant clones for isoleucine and valine biosynthesis (file A) ormethionine biosynthesis (file D) are clearly larger than the plasmidpRO1614 cloning vector (file C). This relationship reflects theacquisition, using recombinant DNA technology of chromosomal DNAdirecting the biosynthesis of the amino acids in question. Therecombinant plasmid maintained carbenicillin resistance as expected.

The novel plasmids of the present invention and RPl have been depositedfor reference purposes with the Northern Regional Research Laboratoryand are freely available upon request by number. The plasmids are alsoavailable from the University of Michigan, Ann Arbor, Mich. by internalreference numblers:

    ______________________________________                                        Internal                                                                      Reference      NRRL Reference                                                 ______________________________________                                        RP1            B-12123                                                        pRORP1/1600    B-12124                                                        pRO1601        B-12125                                                        pRO1613        B-12126                                                        pRO1614        B-12127                                                        pRO1615        B-12149                                                        pRO1616        B-12148                                                        ______________________________________                                    

It should be recognized that there are many experiments that might beperformed which will occur to those skilled in the art. It wil berecognized that there are legal restraints to such experiments and inany event they will be obvious to those skilled in the art.

I claim:
 1. A deoxyribonucleic acid fragment for forming plasmids, thefragment being formed from a first plasmid originally derived from aplasmid aggregation with plasmid RPl, the fragment measuring about 2×10⁶daltons or less in molecular size, wherein the fragment is from pRO1600as carried on Pseudomonas aeruginosa NRRL-B-12124 and includes acritical restriction endonuclease BglI digestion fragment from pRO1600which is indispensable for replication in a plasmid.
 2. The fragment ofclaim 1 wherein the fragment is the BglI digest of pRO1600.
 3. The DNAfragment of claim 1 wherein the fragment of pRO1600 is derived from anendonuclease digestion of pRO1601, pRO1613, PRO1614, pRO1615 or pRO1616as carried in Pseudomonas aeruginosa NRRL-B-12125, NRRL-B-12126,NRRL-B-12127, NRRL-B-12149 and NRRL-B-12148, respectively, which arederived from pRO1600.